METTL3-Mediated m6A Modification Enhances the Function of Adipose-Derived Stem Cells Under Hypoxic Conditions Thereby Improving Fat Graft Retention.

BACKGROUND: Adipose-derived stem cells (ADSCs) have important application prospects in the field of regenerative medicine, such as adjuvant autologous fat transplantation (AFT), due to their multidirectional differentiation and immunomodulatory functions. However, functional limitation of ADSCs in hypoxic environments may affect their effectiveness in clinical applications. Hypoxic preconditioning is a potential strategy to improve the function of ADSCs by enhancing the antioxidant capacity and metabolic adaptations of the cells, but the optimal hypoxic conditions and the mechanism of action have not yet been clarified. METHODS: ADSCs were extracted and pretreated with hypoxia in order to explore its effect on the function of ADSCs. The activity, apoptosis level, proliferation ability, and antioxidant capacity of ADSCs under normoxic and hypoxic conditions were evaluated using flow cytometry (FCM), live-dead cell fluorescence assay, and apoptosis flow assay. Further, the role of METTL3-mediated m6A modification in hypoxic preconditioning was explored by real-time fluorescence quantitative PCR, protein immunoblotting assay, and m6A modification level detection. Finally, the effect of hypoxic preconditioning of ADSCs on fat graft retention was verified by the mouse AFT model. RESULTS: We found that the survival of ADSCs was not affected by 5% O(2) pretreatment. Moreover, the cell viability and proliferation of ADSCs were enhanced after 24 h of anoxic preconditioning. Therefore, we determined that 5% O(2) treatment for 24 h was the best hypoxic pretreatment condition for ADSCs, which enhanced the antioxidant capacity of ADSCs, reduced apoptosis. METTL3-mediated m6A modification played a critical role in hypoxic preconditioning to reduce apoptosis in ADSCs. It was verified in a mouse model that hypoxia preconditioning of ADSCs significantly improved adipose graft retention and promoted neovascularization. CONCLUSIONS: METTL3-mediated modification of m6A enhances the function of ADSCs under hypoxic conditions to improve adipose graft retention. These findings provide a new strategy and theoretical basis for improving the clinical outcome of fat grafting, as well as new molecular targets for future research.